Novel compositions enriched in gamma delta t cells, methods of preparation, and uses thereof

Abstract

Provided herein are novel compositions enriched in gdT cells with high therapeutic potential. Methods to produce such compositions and methods of uses thereof in adoptive immunotherapies are also provided.

1 . A method of manufacturing a cell population enriched in gamma delta T (gdT) cells, comprising culturing a source cell population comprising gdT cells in a medium supplemented with (i) a phosphoantigen, (ii) a cytokine, and (iii) human platelet lysate (“HPL”). 
     
     
         2 . The method of  claim 1 , wherein the cell population is not contacted with a feeder cell or tumor cell during the culture. 
     
     
         3 . The method of  claim 1  that does not include positively selecting for gdT cells. 
     
     
         4 . The method of  claim 1 , wherein the cell population is cultured for 3 to 40 days, 4 to 40 days, 5 to 40 days, 6 to 40 days, 7 to 40 days, 10 to 40 days, 10 to 30 days, 6 to 20 days, 12 to 20 days, or 14 to 18 days. 
     
     
         5 . The method of  claim 1 , further comprising depleting alpha beta T (abT) cells. 
     
     
         6 . The method of  claim 5 , wherein the abT cells are depleted around the half-time of the culture. 
     
     
         7 . The method of  claim 5 , wherein the cells are cultured for 14 to 18 days and the abT cells are depleted between Day 4 and Day 10. 
     
     
         8 . The method of  claim 1 , wherein the cytokine is replenished during the culture. 
     
     
         9 . The method of  claim 8 , wherein the cytokine is replenished once per week, twice per week, three times per week, every other day, or daily. 
     
     
         10 . The method of  claim 1 , wherein the cytokine is interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-33 (IL-33), or any combination thereof. 
     
     
         11 . The method of  claim 10 , wherein the cytokine is IL-2. 
     
     
         12 . The method of  claim 1 , wherein the cytokine is supplemented at a concentration of 200-3000 IU/mL. 
     
     
         13 . The method of  claim 1 , wherein the phosphoantigen is not replenished during the culture. 
     
     
         14 . The method of  claim 1 , wherein the phosphoantigen is a bisphosphonate selected from the group consisting of clodronate, etidronate, alendronate, pamidronate, zoledronate (zoledronic acid), neridronate, ibandronate, and pamidronate. 
     
     
         15 . The method of  claim 14 , wherein the phosphoantigen is zoledronate. 
     
     
         16 . The method of  claim 1 , wherein the phosphoantigen is selected from the group consisting of bromohydrin pyrophosphate (BrHPP), 4-hydroxy-but-2-enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP). 
     
     
         17 . The method of  claim 1 , wherein the phosphoantigen is supplemented at a concentration of 0.1-20 μM. 
     
     
         18 . The method of  claim 1 , wherein the HPL is supplemented at a concentration of 1-20 vol %. 
     
     
         19 . The method of  claim 1 , wherein the medium comprises glucose at a concentration of 600-5000 mg/L. 
     
     
         20 . The method  claim 1 , wherein the medium is a serum-free medium. 
     
     
         21 . The method of  claim 1 , wherein the cell population is cultured in a device containing an air-permeable surface. 
     
     
         22 . The method of  claim 21 , wherein the device is a G-Rex device. 
     
     
         23 . The method of  claim 1 , wherein the source cell population comprises peripheral blood mononuclear cells (PBMCs), bone marrow, umbilical cord blood, or a combination thereof. 
     
     
         24 . The method of  claim 23 , wherein the source cell population comprises PBMCs. 
     
     
         25 . The method of  claim 24 , further comprising obtaining the PBMCs from peripheral blood. 
     
     
         26 . The method of  claim 1 , wherein the gdT cells in the source cell population are expanded for at least 1,000 fold during the culture. 
     
     
         27 . The method of  claim 1 , wherein at least 75% of the resulting cell population are gdT cells. 
     
     
         28 . The method of  claim 1 , further comprising adding a targeting moiety to the surface of the cells in the resulting cell population. 
     
     
         29 . The method of  claim 28 , wherein the targeting moiety is complexed to the cell surface via the interaction between a first linker conjugated to the targeting moiety and a second linker conjugated to the cell surface. 
     
     
         30 . The method of  claim 28 , wherein the targeting moiety is exogenously expressed. 
     
     
         31 . The method of  claim 1 , further comprising cryopreserving the cell population after the culture. 
     
     
         32 . A population of cells obtained by the method of  claim 1 . 
     
     
         33 . A population of cells comprising at least 70% gdT cells, wherein (1) the gdT cells express at least 400 DNAM-1 molecules per cell on average; (2) at least 30% of the gdT cells are CD69 + ; or both (1) and (2). 
     
     
         34 . The population of cells of  claim 33 , wherein the gdT cells express at least 500, at least 1000, at least 2000, or at least 3000 DNAM-1 molecules per cell on average. 
     
     
         35 . The cell population of  claim 33 , wherein at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the gdT cells are CD69+. 
     
     
         36 . The cell population of  claim 33 , wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the gdT cells are terminally differentiated effector (TDEM) cells. 
     
     
         37 . The cell population of  claim 33 , comprising at least 1×10 6 , at least 5×10 6 , at least 1×10 7 , at least 5×10 7 , at least 1×10 8 , at least 5×10 8 , at least 1×10 9 , at least 5×10 9 , at least 1×10 10 , at least 5×10 10 , or at least 1×10 11  gdT cells. 
     
     
         38 . The cell population of  claim 33 , wherein the cell population has not been positively selected for gdT cells. 
     
     
         39 . The cell population of  claim 33 , where the cell population has been cultured for 20 days or less since the source cell population from which the cell population is derived or obtained from a single donor. 
     
     
         40 . The cell population of  claim 33 , wherein:
 (1) the gdT cells express at least 400 CD56 molecules per cell on average;   (2) the gdT cells express at least 400 CD16 molecules per cell on average;   (3) the gdT cells express at least 400 NKG2D molecules per cell on average;   (4) the gdT cells express at least 400 CD107a molecules per cell on average;   (5) the gdT cells express at most 2800 PD-1 molecules per cell on average;   (6) the gdT cells express at least 5000 DNAM-1 molecules per cell on average;   (7) the gdT cells express at least 400 CD69 molecules per cell on average; or   (8) the gdT cells express at least 100 Granzyme B molecules per cell on average;   
       or any combination thereof. 
     
     
         41 . The cell population of  claim 33 , wherein at least 30% of the gdT cells are Vδ2 T cells. 
     
     
         42 . The cell population of  claim 33 , wherein at least 10% of the gdT cells comprise a targeting moiety complexed to the cell surface. 
     
     
         43 . The cell population of  claim 42 , wherein the targeting moiety is not a nucleic acid. 
     
     
         44 . The cell population of  claim 42 , wherein the targeting moiety is an antibody or antigen binding unit that specifically binds to a biological marker on a target cell. 
     
     
         45 . The cell population of  claim 44 , wherein the biological marker is a tumor antigen. 
     
     
         46 . The cell population of  claim 44 , wherein the gdT cells express a chimeric antigen receptor (CAR) or a T cell receptor (TCR) that comprises the antibody or antigen binding fragment. 
     
     
         47 . The cell population of  claim 42 , wherein the targeting moiety is not produced by the gdT cells. 
     
     
         48 . The cell population of  claim 42 , wherein the targeting moiety is complexed to the cell surface via the interaction between a first linker conjugated to the targeting moiety and a second linker conjugated to the cell surface. 
     
     
         49 . The cell population of  claim 48 , wherein the first linker is a first polynucleotide, and the second linker is a second polynucleotide. 
     
     
         50 . The cell population of  claim 49 , wherein (1) the first polynucleotide has 4 to 500 nucleotides, (2) the second polynucleotide has 4 to 500 nucleotides, or both (1) and (2). 
     
     
         51 . The cell population of  claim 32  that is cryopreserved. 
     
     
         52 . A pharmaceutical composition comprising the cell population of  claim 32  and a pharmaceutically acceptable carrier. 
     
     
         53 . The cell population of  claim 32 , that can maintain its therapeutic potency after being stored at or below 0° C. for at least one week, at least two weeks, at least 1 month, at least 3 months, or at least 6 months. 
     
     
         54 . Use of the cell population or the pharmaceutical composition of  claim 32  in an adoptive immunotherapy. 
     
     
         55 . Use of the cell population or the pharmaceutical composition of  claim 32  in the treatment of a disease or disorder. 
     
     
         56 . A method of treating a disease or disorder in a subject in need thereof, comprising administering the cell population or the pharmaceutical composition of  claim 32  to the subject. 
     
     
         57 . The use of  claim 55 , wherein the disease or disorder is tumor or cancer. 
     
     
         58 . The use of  claim 55 , wherein the disease or disorder is an autoimmune disease, a neuronal disease, a hematopoietic cell-related disease, metabolic syndrome, a pathogenic disease, HIV or other viral infection, fungal infection, protozoan infection, or bacterial infection. 
     
     
         59 . The method of  claim 56 , wherein the subject is human.